The Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid. Since its isolation more than 100 years ago, Streptococcus pneumoniae has been one of the more intensively studied microbes. For example, much of our early understanding that DNA is, in fact, the genetic material was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe. Despite the vast amount of research with Streptococcus pneumoniae, many questions concerning the virulence of this microbe remain. It is particularly preferred to employ Streptococcal genes and gene products as targets for the development of antibiotics.
The frequency of Streptococcus pneumoniae infections has risen dramatically in the past few decades. This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems. It is no longer uncommon to isolate Streptococcus pneumoniae strains which are resistant to some or all of the standard antibiotics. This phenomenon has created an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism.
Moreover, the drug discovery process is currently undergoing a fundamental revolution as it embraces “functional genomics” that is, high throughput genome- or gene-based biology. This approach is rapidly superseding earlier approaches based on “positional cloning” and other methods. Functional genomics relies heavily on the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available as well as from other sources. There is a continuing and significant need to identify and characterize further genes and other polynucleotides sequences and their related polypeptides, as targets for drug discovery.
Clearly, there exists a need for polynucleotides and polypeptides, such as the pth embodiments of the invention, that have a present benefit of, among other things, being useful to screen compounds for antibiotic activity. Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease. There is also a need for identification and characterization of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease.
Peptidyl tRNA hydrolase plays a pivotal role in mRNA translation, regenerating tRNA from peptidyl tRNA intermediates. Pth activity is apparently ubiquitous and pth homologs have been identified in a wide range of bacteria. Analogous activity in mammals is mediated by a quite different set of enzymes. Inhibition of Pth activity in E. coli results in the inhibition of protein synthesis and ultimately cell death. We have shown that Pth is essential for in vitro growth in S. pneumoniae and S. aureus and that the gene encoding for this protein is expressed during infection of both pathogenic strains. Therefore, inhibitors of this protein could prevent the bacterium from establishing or maintaining infection of the host and thereby have utility in anti-bacterial therapy.
Certain of the polypeptides of the invention possess significant amino acid sequence homology to a known B. subtilis pth protein. (Dutka S, et al., Role of the 1–72 base pair in tRNAs for the activity of Escherichia coli peptidyl-tRNA hydrolase. Nucleic Acids Res 1993 Aug. 25;21(17):4025–4030; Heurgue-Hamard V, et al., The growth defect in Escherichia coli deficient in peptidyl-tRNA hydrolase is due to starvation for Lys-tRNA(Lys). EMBO J 1996 Jun. 3;15(11):2826–2833; Schmitt E, et al., Crystal structure at 1.2 A resolution and active site mapping of Escherichia coli peptidyl-tRNA hydrolase. EMBO J 1997 Aug. 1;16(15):4760–4769; Swiss-prot Accession number: P37470 Peptidyl tRNA hydrolase (pth) B. subtilis; Ogasawara, N., et al. Systematic sequencing of the 180 kilobase region of the Bacillus subtilis chromosome containing the replication origin; DNA Res. 1 (1), 1–14 (1994); TIGR contig 4125.)